A spectroscopic study of phenylbutazone and aspirin bound to serum albumin in rheumatoid diseases.

Interaction of phenylbutazone (PBZ) and aspirin (ASA), two drugs recommended in rheumatoid diseases (RDs), when binding to human (HSA) and bovine (BSA) serum albumins, has been studied by quenching of fluorescence and proton nuclear magnetic resonance ((1)HNMR) techniques. On the basis of spectrofluorescence measurements high affinity binding sites of PBZ and ASA on albumin as well as their interaction within the binding sites were described. A low affinity binding site has been studied by proton nuclear magnetic resonance spectroscopy. Using fluorescence spectroscopy the location of binding site in serum albumin (SA) for PBZ and ASA was found. Association constants K(a) were determined for binary (i.e. PBZ-SA and ASA-SA) and ternary complexes (i.e. PBZ-[ASA]-SA and ASA-[PBZ]-SA). PBZ and ASA change the affinity of each other to the binding site in serum albumin (SA). The presence of ASA causes the increase of association constants K(aI) of PBZ-SA complex. Similarly, PBZ influences K(aI) of ASA-SA complex. This phenomenon shows that the strength of binding and the stability of the complexes increase in the presence of the second drug. The decrease of K(aII) values suggests that the competition between PBZ and ASA in binding to serum albumin in the second class of binding sites occurs. The analysis of (1)HNMR spectral parameters i.e. changes of chemical shifts and relaxation times of the drug indicate that the presence of ASA weakens the interaction of PBZ with albumin. Similarly PBZ weakens the interaction of ASA with albumin. This conclusion points to the necessity of using a monitoring therapy owning to the possible increase of uncontrolled toxic effects.

The spectroscopic studies of humic acid extracted from sediment collected at different seasons.

Goczalkowice Reservoir is the biggest water reservoir in the south of Poland. For our studies bottom sediments were collected from eight different places of the reservoir at various seasons of the year. EPR spectroscopy was applied to analyse both quantitatively and qualitatively the humic acids (HA) present in the samples. EPR spectra of the extracted HA exhibited broad lines from the paramagnetic metal ions and narrow lines from free radicals. The values of the free radical concentration obtained for HA amounted to 1.14-13.6 × 1016 spin g(-1) depending on the season and the place of sample collection. The values of the g factor obtained for HA were of the range 2.0027-2.0035. The EPR studies show that HA extracted from bottom sediment collected at various points of the Goczalkowice Reservoir exhibit similar physical-chemical properties. It was also observed that the depth of the reservoir affects the content of the oxygen functional groups as well as the free radical concentration in HA. The identification of the functional groups was done by means of IR. UV/VIS spectroscopy was used to estimate the maturity of the HA.

Influence of myristic acid on furosemide binding to bovine serum albumin. Comparison with furosemide-human serum albumin complex.

Fluorescence studies on furosemide (FUR) binding to bovine serum albumin (BSA) showed the existence of three or four binding sites in the tertiary structure of the protein. Two of them are located in subdomain IIA, while the others in subdomains IB and/or IIIA. Furosemide binding in subdomain IB is postulated on the basis of run of Stern-Volmer plot indicating the existence of two populations of tryptophans involved in the interaction with FUR. In turn, the significant participation of tyrosil residues in complex formation leads to the consideration of the subdomain IIIA as furosemide low-affinity binding site. The effect of increasing concentration of fatty acid on FUR binding in all studied binding sites was also investigated and compared with the previous results obtained for human serum albumin (HSA). For BSA the lesser impact of fatty acid on affinity between drug and albumin was observed. This is probably a result of more significant role of tyrosines in the complex formation and different polarity of microenvironment of the fluorophores when compared HSA and BSA. The most distinct differences between FUR-BSA and FUR-HSA binding parameters are observed when third fatty acid molecule is bound with the protein and rotation of domains I and II occurs. However these structural changes mostly affect FUR low affinity binding sites.

Alterations of furosemide binding to serum albumin induced by increased level of fatty acid.

Localization of high and low affinity binding sites of furosemide in human serum albumin (HSA) as well as the influence of myristic acid on the drug binding to the albumin using fluorescence quenching method was investigated. Two independent classes of binding site in subdomain IIA of HSA structure were found. Alteration of protein affinity towards the drug and the participation of tryptophanyl and tyrosil residues in drug-albumin interaction for the determined binding sites were studied. It was concluded that association of myristic acid in its low affinity binding sites which corresponds to elevated fatty acid level in vivo, significantly decreases albumin affinity towards furosemide.

The influence of dietary habits and pathological conditions on the binding of theophylline to serum albumin.

The influence of fatty acids (FA) on theophylline (Th) binding to human serum albumin (HSA) in its high and low affinity binding sites was investigated. The content of studied FA solutions corresponds to the ones associating with different dietary habits and pathological states in vivo. Using fluorescence and (1)H NMR spectroscopy two high and two low affinity binding sites of Th in HSA structure were found. For each site several binding parameters in the absence and presence of FA were estimated. The results showed that the impact of FA on the affinity of HSA towards Th in high affinity binding sites is negligible whereas binding of the drug in low affinity sites decreases significantly in the presence of FA. It was observed that this effect is dependent on the number of fatty acid molecules bound to the protein while the chemical structure of fatty acids contained in the solution plays a minor role.

Binding of 6-propyl-2-thiouracil to human serum albumin destabilized by chemical denaturants.

We compared the binding affinity of 6-propyl-2-thiouracil (PTU) with native and destabilized human serum albumin (HSA) as a model to assess the binding ability of albumin in patients suffering from chronic liver or renal diseases. Urea (U) and guanidine hydrochloride (Gu.HCl) at a concentration of 3.0M were used as denaturation agents. Increasing the concentration of PTU from 0.8x10(-5) to 1.20x10(-4)M in the systems with HSA causes a decrease in fluorescence intensity of the protein excited with both 280 and 295nm wavelengths. The results indicate that urea and Gu.HCl bind to the carbonyl group and then to the NH-group. To determine binding constants we used the Scatchard plots. The presence of two classes of HSA-PTU binding sites was observed. The binding constants (K(b)) are equal to 1.99x10(4)M(-1) and 1.50x10(4)M(-1) at lambda(ex)=280nm, 5.20x10(4)M(-1) and 1.65x10(4)M(-1) at lambda(ex)=295nm. At lambda(ex)=280nm the number of drug molecules per protein molecule is a(I)=1.45 and a(II)=1.32 for I and II binding sites, respectively. At lambda(ex)=295nm they are a(I)=0.63 and a(II)=1.54 for the I and II binding sites. The estimation of the binding ability of changed albumin in the uremic and diabetic patients suffering from chronic liver or renal diseases is very important for safety and effective therapy.

Interaction of phenylbutazone and colchicine in binding to serum albumin in rheumatoid therapy: 1H NMR study.

The monitoring of drug concentration in blood serum is necessary in multi-drug therapy. Mechanism of drug binding with serum albumin (SA) is one of the most important factors which determine drug concentration and its transport to the destination tissues. In rheumatoid diseases drugs which can induce various adverse effects are commonly used in combination therapy. Such proceeding may result in the enhancement of those side effects due to drug interaction. Interaction of phenylbutazone and colchicine in binding to serum albumin and competition between them in gout has been studied by proton nuclear magnetic resonance ((1)H NMR) technique. The aim of the study was to determine the low affinity binding sites, the strength and kind of interaction between serum albumin and drugs used in combination therapy. The study of competition between phenylbutazone and colchicine in binding to serum albumin points to the change of their affinity to serum albumin in the ternary systems. This should be taken into account in multi-drug therapy. This work is a subsequent part of the spectroscopic study on Phe-COL-SA interactions [A. Sulkowska, et al., J. Mol. Struct. 881 (2008) 97-106].

Comparison of humification processes occurring during sewage purification in treatment plants with different technological processes.

The course of the humification process of sewage sludge collected from three biologic-mechanical treatment plants with different treatment technologies was studied. The maturity of sewage sludge and its usefulness for agricultural purposes was also discussed. The physical-chemical properties of humic acids extracted from sewage sludge received from comparable stages of sludge purification were described. Changes of the sludge properties during sewage purification and the progress of the humification process were investigated with EPR, IR and UV/VIS spectroscopic methods. The content of the elements and the carboxylic groups in humic acids extracted from each stage of the sewage treatment were also determined. It was found that the humification processes take place in all three treatment plants but with different intensities resulting from the differences in the individual cleaning processes in these plants. The most intensive changes of physical-chemical parameters in the extracted humic acids were observed in the anaerobic digester where mesophilic fermentation occurs. The sludge oxygenation processes also significantly affect the course of the humification process during sewage treatment.

NMR study of the humification process during sewage sludge treatment.

Humic acids, extracted from sludge at the biologic-mechanical sewage treatment plant in Jastrzebie Zdroj, have been investigated by means of (1)H, (13)C and (31)P NMR spectroscopy. Sludge samples for studies were taken from the primary settling tank, the nitrification chamber, the digestion chamber and the sludge drying beds. The (1)H NMR analysis of humic acid extracted from sludge at various stages of sewage treatment confirmed the presence of the functional groups that are characteristic for humic substances, and the analysis showed changes in their relative intensities. The (13)C NMR indicated that the aromatisation of the humic acid increased during sewage treatment. Moreover, the analysis of the (31)P NMR spectra allowed us to observe the changes in the phosphorus groups of the studied humic acids.

Changes of serum albumin affinity for aspirin induced by fatty acid.

Saturated fatty acids such as myristic acid play an important role in the pathogenesis of cardiovascular disorders. Using the quenching fluorescence method we examined the influence of myristate on the changes of transporting protein affinity towards aspirin-the most popular anticoagulant. Our results showed that the presence of the myristic acid alters the stability of the anticoagulant-albumin complex. The ranges of [myristate]/[albumin] molar ratio at which the stability of drug-protein complex increases or decreases were determined. The differences in interaction between ligands and human or bovine serum albumins were identified. The competition in binding of ligands with these albumins was also described.

Primary and secondary relaxations in bis-5-hydroxypentylphthalate.

Broadband dielectric spectroscopy was used to study the relaxation dynamics in bis-5-hydroxypentylphthalate (BHPP) under both isobaric and isothermal conditions. The relaxation dynamics exhibit complex behavior, arising from hydrogen bonding in the BHPP. At ambient pressure above the glass transition temperature T(g), the dielectric spectrum shows a broad structural relaxation peak with a prominent excess wing toward higher frequencies. As temperature is decreased below T(g), the excess wing transforms into two distinct peaks, both having Arrhenius behavior with activation energies equal to 58.8 and 32.6 kJmol for slower (beta) and faster (gamma) processes, respectively. Furthermore, the relaxation times for the beta process increase with increasing pressure, whereas the faster gamma relaxation is practically insensitive to pressure changes. Analysis of the properties of these secondary relaxations suggests that the beta peak can be identified as an intermolecular Johari-Goldstein (JG) process. However, its separation in frequency from the alpha relaxation, and both its activation energy and activation volume, differ substantially from values calculated from the breadth of the structural relaxation peak. Thus, the dynamics of BHPP appear to be an exception to the usual correlation between the respective properties of the structural and the JG secondary relaxations.

Effect of pressure on dynamic heterogeneity in dendrimeric alkyd resin.

Broadband dielectric spectroscopy is employed to investigate the non-Debye relaxation behavior in a dendrimeric alkyd resin. From temperature-dependent measurements at ambient pressure, we found a very broad distribution of relaxation times. This is attributed to the complex geometrical topology of the molecule. However, compression significantly reduces the non-Debye character of the dielectric response; thus, pressure induces dynamic homogeneity in the dendrimeric alkyd resin.

Fluidlike behavior of dielectric permittivity in a wide range of temperature above and below the nematic-isotropic transition.

Singular behavior of the static dielectric permittivity of n-alkyloxycyanobiphenyls (CnH2n+1O-Ph-Ph-C [triple bond] N, n=6, 7) was studied above and below the nematic clearing point (T(I-N)). On approaching the clearing point, the evolution of principal components of the nematic permittivity tensor, epsilon(parallel) and epsilon(perpendicular), is described by the order parameter exponent beta approximately 0.25. The mean value of the nematic permittivity epsilon(mean)=(epsilon(parallel)+2epsilon(perpendicular))/3 exhibits a singular behavior similar to that observed in the isotropic phase and that for the diameter of the coexistence curve in binary mixtures. The derivative of experimental data d(epsilon)iso(T)/dT and d(epsilon)mean(T)/dT shows the specific-heat-like behavior with universal exponents alpha=alpha' approximately 0.5. Results obtained confirm the hypothesis of the fluidlike, pseudospinodal, and tricritical behavior of the isotropic to nematic phase transition. [A. Drozd-Rzoska, Phys. Rev. E 59, 5556 (1999)].

Low-frequency dielectric relaxation in rubber.

A rubber sample is investigated by dielectric spectroscopy in the frequency range from 10(-2)

[Proposals of rational procedures for certifying occupational voice disorders]. / Propozycje racjonalnego postepowania w orzekaniu o chorobie zawodowej narzadu glosu.

Following a long period of experience acquired in assessing occupation-related voice disorders, the proposals of objective measures, facilitating the certification of occupational disease of the voice organ in accordance with the binding legal regulations, are presented. The importance of documentary evidence of at least two years of treatment and phoniatric rehabilitation is stressed. A need to prevent occupation-related voice disorders through phoniatric diagnostic procedures is highlighted.

Objective evidence for tinnitus from spontaneous emission variability.

Noise exposure is the most common cause for the generation of tinnitus. This study evaluated the variability of spontaneous emissions in industrial workers exposed to noise and reporting the presence of tinnitus in comparison with those exposed to noise but without tinnitus. The assumption being that exposure to noise leads to some instability within the cochlea, which alters the spontaneous emission activity. Thus those experiencing tinnitus may show greater variability than those without tinnitus. 198 mill workers in Poland exposed to noise levels between 85-95dBA for a mean of 12+/-6.6 years, 104 of whom had reported the presence tinnitus and 94 without tinnitus were evaluated for otoscopy, audiometry and otoacoustic emissions. The tests were repeated between 5-10 days in most subjects to check for variability. There were significant differences in the mean age, pure tone average, transient emissions amplitude and variability between groups with and without tinnitus. There were no significant differences between sessions for these measures in either group. Those with tinnitus had poorer thresholds by an average of around 15dB, and reduced TOAE of around 2.6dB compared with those without tinnitus. There are a number of factors such as age, pure tone thresholds and tinntius, which may be responsible for the reduction in emissions. For the purposes of examining SOAE stability, all SOAE peaks were classed as stable if SOAE frequency in the two sessions remained unchanged and variable if SOAE peaks were present in both sessions but shifted in frequency or present in one sessions and absent in the other. SOAE were present in 73.1% of tinnitus group and in 50% of non-tinnitus group. Of these 92% of the tinnitus group had present and variable SOAE whereas 48.9% of the non-tinnitus group did. Thus the positive predictive value was calculated at 65% for those with variable SOAE having tinnitus and significantly higher at 86% negative predictive value for those with stable SOAE having no tinntus. The likelihood ratio of tinnitus being present given that SOAE are present and variable is 1.87 and is significantly reduced for no tinnitus given that SOAE are present and stable at 0.156. This study has clearly demonstrated that the incidence of spontaneous emissions is higher in noise-exposed workers than previously observed and the stability from week to week is significantly lower in those with subjective tinnitus.

[Tinnitus in noise-induced hearing impairment]. / Szumy uszne w uszkodzeniach sluchu spowodowanych halasem.

The analysis of the epidemiological data indicates that exposure to noise is widespread and it is one of the most common causes of tinnitus, estimated at about 20.7% according to Hazell; 28% according to Axelsson; and 42% according to Palmer. Bearing in mind the scantiness of reliable data on the incidence and nature of tinnitus in persons exposed to industrial noise, and especially the need for the objectivity of this subjective symptom, the authors have decided to undertake the study aimed at assessing the interrelation between tinnitus, the magnitude and kind of hearing impairment, and otoacoustic emission. The study group included 191 persons aged 42.5 +/- 7.6 years (range, 25 to 65), occupationally exposed to noise at the levels of 88-92 dB(A) for 26.9 +/- 4.6 years (range, 9 to 30) who had reported hearing disorders and tinnitus. The control group, matched by similar age and duration of employment, consisted of 80 persons with perceptive hearing impairment induced by industrial noise who had not complained of tinnitus. The results of the study revealed that in 59.7% of the study subjects, noise proved to be one of the most probable factors responsible for the development of tinnitus. The presence of tinnitus was found in 22.5% and in 46% of the study subjects after 10 years and 11-20 years of noise exposure, respectively. In 95.8% of workers, tinnitus was associated with hearing loss, and only in 4.2% of cases it occurred in ears with normal hearing. In persons exposed to noise, tinnitus was most frequently (59.2%) bilateral and permanent. Following the audiologic examinations, verified by objective audiometry (tympanometry, ABR), cochlear hearing impairment was found in 68.6%; retrochochlear in 8.37%; mixed and other forms of impairment, e.g. presbyacousis, in 19.4% of subjects. The audiologic assessment of tinnitus demonstrated that in 62.3% of persons, tinnitus occurred at high frequencies and correlated with the magnitude of hearing impairment in the tonal audiogram. The tinnitus intensity ranged between 10-15 dB and 45 dB. In 40.3% of those under study, noise was not the only tinnitus-risk factor. In this group of persons, the presence of predisposing diseases was also observed, e.g. hypertension, diabetes, atherosclerosis, disturbed lipid metabolism and other etiologic factors that might have impact on the tinnitus incidence, ototoxic drugs, for example. The measurements of evoked otoacoustic emission (EOAE and DPOAE) revealed in 58.63% of persons significant differences (p < 0.01) in the amplitude and spectrum of EOAE in the ears with tinnitus as compared to the ears without tinnitus with a similar hearing threshold. Whereas in 27.74% of subjects, no differences in the EOAE measurements in the ears with or without tinnitus were observed. The results of DPOAE measurements showed in 62% of subjects significant differences in DP-grams in the ears with tinnitus as compared to the ears without tinnitus (p < 0.01). Interestingly, the differences in measurements of both types of evoked emissions (EOAE and DPOAE), expressed by the lowered amplitude, narrowed spectrum, reduction of emission or its complete fading in a limited area of high frequencies, were demonstrated in the ears with tinnitus only in retrocochlear hearing impairment, as compared to those free from tinnitus. The evaluation of the EOAE and DPOAE measurements seems to prove that this method may be useful in assessing the contribution of the cochlear mechanisms to the incidence of tinnitus and in distinguishing between tinnitus generated in cochlea and tinnitus with the source at other levels of the hearing organ or beyond it. Our study failed to determine the interrelation between tinnitus and spontaneous emissions as the emission was registered only in about 12% of persons exposed to noise, including 2% of those with normal hearing.

[Nasal lavage, rhinomanometry and rhinoscopy in diagnosing occupational airway allergy]. / Badanie popluczyn nosowych oraz badanie rynomanometryczne i badanie rynologiczne w diagnostyce zawodowych alergoz ukladu oddechowego.

The aim of the study was to estimate the usefulness of nasal lavage, rhinomanometry and rhynoscopy in diagnosing occupational allergic rhinitis and bronchial asthma. 26 subjects with suspected bronchial asthma and allergic rhinitis due to occupational allergens were examined. Each subject had medical history collected and underwent physical examination, skin prick tests (SPT) with common and occupational allergens, determination of total and specific IgE, specific bronchial or nasal provocation with determination of spirometric parameters, morphological and biochemical changes in nasal lavage fluid (NLF). Moreover the anterior rhinomanometry and rhynoscopy were performed before and after the provocation. A significant increase in the percentage of eosinophils and albumin was observed in NLF up till 24 hr after the specific challenge, but only in the group of 16 subjects with diagnosed occupational airway allergy. The authors observed also the presence of mucosal oedema and rhinorrhea in this group of patients more frequently than in the group of patients without diagnosed occupational airway disease. No significant changes were observed in the frequency of positive rhinomanometry test between the analysed groups of patients.

[Assessment of the hearing system in workers chronically exposed to carbon disulfide and noise]. / Ocena stanu sluchu u pracowników narazonych na przewlekle dzialanie dwusiarczku wegla i halasu.

Epidemiological studies carried out in the years 1984-1993 revealed carbon disulfide (CS2) to be one of the major chemical occupational hazards. Whereas among physical factors, noise was found to be the most common threat. Industrial workers very often face a combined exposure of two or more factors, like CS2 and noise, responsible for significant biological risk to the human health, although health effects of such an exposure have not as yet been thoroughly recognized and explored. The aim of this study was to assess changes in the hearing system in people exposed to toxic effect of CS2, recognized as characteristic of an overall clinical history of chronic poisoning by this solvent, in cases of concomitant exposure to noise exceeding maximum allowable levels. The study covered 80 workers aged 44-65 years (mean = 44.9 +/- 5.1), employed in a spinning mill of viscose fibre for 20.3 years on average (+/- 5.4 years) with clinically observed chronic CS2 poisoning, and a group of 40 people (mean age = 56.8 years) exposed to CS2 but without subjective or objective symptoms indicating chronic poisoning by the solvent. Both groups of subjects at similar age and with almost the same duration of employment were exposed to CS2 in concentrations changing in time between 10 and 35 mg/m3 (mean concentration = 25.8 mg/m3), and to continuous noise with the level ranging from 88 to 92 dB(A) for six hrs per one shift. The control group was composed of 40 workers (mean age = 52.0 +/- 5.3 years) employed in the cotton industry plant, without contact with CS2 or other chemicals and working in the acoustic environment with similar level of exposure to noise (86-93 dB(A)). Audiological and electronystagmographic examinations revealed bilateral retrocochlear hearing impairment associated with symptoms of the central vestibular syndrome in 97.5 subjects with diagnosed chronic CS2 poisoning. In workers free from clinical symptoms of chronic CS2 poisoning, perceptive hearing impairment of various degrees was found, including retrocochlear in 45% of subjects and cochlear in 32.5%, while in 22.5% of those under study normal hearing was observed. In the control group of subjects exposed to noise without contact with CS2, sensorineural cochlear hearing loss, typical of chronic acoustic trauma without concomitant vestibular disorders, was revealed. The results of the study show that in subjects with diagnosed chronic CS2 poisoning and exposed to noise, hearing impairment and vestibular disorders occur in the form of central changes which suggests a dominating CS2 toxic effect on the hearing system. But in some people exposed to both CS2 and noise, the hearing impairment in localised in cochlea like in acoustic trauma damage. This probably depends on individual susceptibility to harmful effect of these factors.